Development and Reproduction of Ladybeetles (Coleoptera: Coccinellidae) on the Citrus Aphids Aphis spiraecola Patch and Toxoptera citricida (Kirkaldy) (Homoptera: Aphididae)

Biological Control 18, 287–297 (2000) doi:10.1006/bcon.2000.0833, available online at http://www.idealibrary.com on

Development and Reproduction of Ladybeetles (Coleoptera: Coccinellidae) on the Citrus Aphids Aphis spiraecola Patch and Toxoptera citricida (Kirkaldy) (Homoptera: Aphididae) J. P. Michaud Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850 Received November 1, 1999; accepted February 28, 2000

INTRODUCTION Seven species of Coccinellidae inhabiting citrus groves in Florida were evaluated for ability to develop and reproduce on the citrus aphids Toxoptera citricida (Kirkaldy) and Aphis spiraecola Patch. Choice tests performed with adults and larvae indicated that both aphid species were generally acceptable prey. Coccinella septempunctata L., Coleomegilla maculata fuscilabris (Mulsant), Coelophora inaequalis F., and Olla v-nigrum Mulsant were unable to complete development on either aphid. Hippodamia convergens Guerin larvae completed development on A. spiraecola with 68% survival; none survived on T. citricida. Only Cycloneda sanguinea (L.) and Harmonia axyridis Pallas completed development on both A. spiraecola (60 and 70% survival, respectively) and T. citricida (100 and 95% survival, respectively). Larval developmental time was shorter on T. citricida than on A. spiraecola, and resulting adults were heavier, differences being more pronounced in H. axyridis. Females of C. septempunctata, C. inaequalis, and O. v-nigrum produced viable eggs on T. citricida. O. v-nigrum did not produce eggs on A. spiraecola. Females of C. m. fuscilabris and H. axyridis produced no eggs on A. spiraecola and mostly infertile eggs on T. citricida. Female H. convergens laid twice as many eggs feeding on T. citricida as on A. spiraecola and egg viability was similar. C. sanguinea females laid similar numbers of eggs on both aphids, but eggs produced on A. spiraecola had higher fertility. Pollen added to the T. citricida diet temporarily improved the fertility of H. axyridis females, but not that of C. sanguinea females. C. sanguinea was judged the best candidate for augmentative biocontrol of T. citricida, the primary vector of citrus tristeza virus. © 2000 Academic Press Key Words: Aphis spiraecola; Coccinella septempunctata; Coelophora inaequalis; Coleomegilla maculata fuscilabris; Cycloneda sanguinea; Harmonia axyridis; Hippodamia convergens; Olla v-nigrum; Toxoptera citricida.

The introduction of the brown citrus aphid (BCA), Toxoptera citricida (Kirkaldy), into southeast Florida in 1995 marked a significant change in the invertebrate fauna associated with citrus in the state (Halbert, 1997; Michaud, 1998). The green citrus aphid (GCA), Aphis spiraecola Patch, has been present in Florida citrus since at least 1923 (Miller, 1929). Although heavy GCA populations cause direct damage to young citrus trees (including leaf distortion and shortening of terminal internodes), BCA is considered a more serious pest because of its efficiency in vectoring citrus tristeza virus (Balaraman and Ramakrishnan, 1979; Yokomi and Damsteegt, 1991). In 1997 and 1998 BCA began to surpass GCA as the dominant aphid in Florida citrus (J. P. Michaud, unpublished). Both aphid species feed on newly flushed citrus leaves, and mixed colonies of the two are common (Michaud, 1999), as are colonies of GCA and Aphis gossypii Glover (J. P. Michaud, unpublished). Because new flush is available only on an intermittent basis in commercial groves, citrus aphids often occur together in time and space and tend to be attacked by the same guild of parasitoids and predators. Parasitism is a relatively minor source of mortality for both GCA and BCA. Lysiphlebus testaceipes Cresson (Hymenoptera: Aphidiidae) is the only primary parasitoid of aphids currently present in Florida citrus (Michaud, 1999). In Florida, its primary hosts are A. gossypii and Toxoptera aurantii (Boyer de Fonscolomb), and neither of these species are abundant in citrus habitats. L. testaceipes readily attacks BCA, and larvae usually develop to the mummy stage, but few adult wasps emerge (Yokomi and Tang, 1996; Michaud and Browning, 1999). Although L. testaceipes may sometimes enter citrus groves in large numbers, elimination of BCA colonies due to parasitism is rare (Michaud, 1999). The GCA is also attacked by L. testaceipes, although the parasitoid does not develop as far

287

1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

288

J. P. MICHAUD

as the mummy stage in Florida. Notwithstanding, L. testaceipes does mummify on GCA in Puerto Rico and the Dominican Republic (J. P. Michaud, unpublished) and in Spain (Michelena and Sanchez, 1997). Biological control of citrus aphid populations is provided primarily by a complex of coccinellid and syrphid species (Michaud, 1999; Michaud and Browning, 1999). Whereas the contributions of these two major groups vary seasonally, comparative field data indicate that coccinellids are the most efficient predatory group (i.e., they are more effective in eliminating BCA colonies prior to the production of the alatae that are instrumental in disease transmission) (Michaud, 1999). These generalist predators can be found feeding as larvae and adults on all species of citrus aphids, whether in pure or mixed colonies. Many larval predators of BCA were reared to adult stages on diets of either BCA or GCA for purposes of identification by Michaud and Browning (1999). Whereas the survival of syrphid larvae was high on both aphid species, that of coccinellid larvae was more variable. Studies by others have suggested that BCA might be unsuitable prey for some coccinellids (Tao and Chiu, 1971; Morales and Burandt, 1985). GCA is possibly a more reliable source of food for coccinellids in citrus than is BCA due to frequent recolonization events. For example, GCA tends to undergo spring population expansion in citrus before BCA, possibly due to early colonization of flush by GCA migrants from alternate hosts not shared by BCA (Komazaki, 1988; J. P. Michaud, unpublished). Whereas BCA can occur at much higher densities than GCA in citrus, it may also remain at very low densities for extended periods. It follows that those coccinellid species that can effectively utilize both aphid species would be best able to maintain populations within citrus that provide continuous aphid control. The present work was undertaken to evaluate and compare the performance of various coccinellids on these two citrus aphids in the hope of identifying species with the greatest potential for biological control of these citrus aphids, primarily BCA. In this study, elements of both the functional response and the numerical response were evaluated for seven species of coccinellid that recruit naturally to aphid colonies in citrus groves: Coccinella septempunctata L., Coelophora inaequalis (F.), Coleomegilla maculata fuscilabris (Mulsant), Cycloneda sanguinea (L.), Harmonia axyridis Pallas, Hippodamia convergens Guerin, and Olla v-nigrum Mulsant. Choice experiments were performed to compare the behavioral responses of larvae and adults to BCA and GCA. Each coccinellid species was tested for ability to complete larval development on both aphid species, and species capable of development were further evaluated for ability to reproduce on the same diets.

MATERIALS AND METHODS

Developmental Assays Stock colonies of T. citricida and A. spiraecola were reared in a greenhouse on Carrizo citrange (Poncirus trifoliata (L.) Osbeck ⫻ Citrus sinensis (L.) Rafinesque) and Viburnum odoratissimum Kerr-Gawl, respectively. Coccinellid eggs were held in plastic petri dishes (5.5 cm diameter ⫻ 1.0 cm height) in a plexiglas terrarium under soft white fluorescent light (14:10 (L:D) h) at 23 ⫾ 2°C and 60 – 65% RH. Preliminary trials indicated that a high proportion of the eggs of most species hatched successfully under these conditions. Individual larvae were placed into numbered plastic petri dishes (as above) within several hours of hatching and randomly divided into two groups of 20, one to receive T. citricida and the other, A. spiraecola. Each larva was provisioned with abundant aphids on a fresh piece of excised plant tissue; dishes were cleaned and fresh plant material and aphids were provided daily until larvae either pupated or died. Experiments were performed in a greenhouse under supplementary metal halide lighting (14:10 (L:D) h) at a mean temperature of 24 ⫾ 2°C. Relative humidity ranged from 60 to 75% throughout trials. Pupae were held at 23 ⫾ 2°C and 60 – 65% RH. Dates of pupation, mortality, and adult emergence were noted. A second colony of A. spiraecola was established on potted sweet orange, C. sinensis, to test whether these aphids would differ in nutritional value to H. convergens larvae compared with those reared on V. odoratissimum. An additional developmental assay was performed with C. m. fuscilabris to determine whether addition of pollen to the diet would improve larval survival on either aphid species. Newly hatched larvae were divided into three treatment groups: one group (n ⫽ 20) received T. citricida ⫹ pollen of the ice plant, Malephora crocea (Jacquin); another received A. spiraecola ⫹ pollen; the third group received pollen alone on a clean citrus leaf plus a moist cotton wick for water. When five or more adult beetles emerged in each treatment, these were dried in an oven at 50°C for 48 h and then weighed on an analytical balance. Data were analyzed by one-way ANOVA (SPSS, 1998). Choice Tests Predators were placed into individual plastic petri dishes (5.5 cm diameter ⫻ 1.0 cm height), each containing 12 GCA and 12 BCA without plant material. Care was taken to ensure that similar-sized aphids were provided (late second instar T. citricida and early fourth instar A. spiraecola) so that predators would not select prey on the basis of their size. All larvae were tested within several hours of eclosion without any prior experience with aphid prey. Adult coccinellids were either field collected (C. septempunctata, C. san-

289

DEVELOPMENT AND REPRODUCTION OF LADYBEETLES

TABLE 1 Survival Rates for Larvae of Seven Coccinellid Species and Mean Dry Weight of Eclosing Adults Percentage larvae surviving to adult

Mean adult weight (⫾SEM) (mg)

GCA

BCA

P

GCA

BCA

5.0 0.0 0.0 60.0 70.0 68. 0.0

0.0 0.0 45.0 100.0 95.0 0.0 0.0

ns a — 0.01 b 0.001 a ns a 0.001 b —

— — — 2.6 ⫾ 0.18 4.9 ⫾ 0.17 3.1 ⫾ 0.15 —

— — — 3.3 ⫾ 0.15 9.7 ⫾ 0.24

C. septempunctata C. inaequalis C. maculata fuscilabris C. s. sanguinea H. axyridis H. convergens O. v-nigrum

Artificial diet

P

11.6 ⫾ 0.62

— — — 0.001 a 0.001 a 0.001 a

—

Note. Larvae (n ⫽ 20) were fed from eclosion on either A. spiraecola (GCA) or T. citricida (BCA). Groups that failed a Levine test for equality of variances were compared using a Mann–Whitney U test. a Significant differences based on one-way ANOVA; ns indicates no significant difference. b Significant differences based on Mann–Whitney U test; ns indicates no significant difference.

guinea, and H. axyridis) or transferred from artificial diet (C. m. fuscilabris and H. convergens). All choice tests were conducted on a laboratory bench under fluorescent light and were repeated with both a white and a black background. This was done to control for possible differences in background contrast between the aphid species that could possibly influence a predator’s rate of encounter and attack. Testing times were varied among species (larvae: 18 –24 h, adults: 1–5 h) so that the period was long enough to permit consumption of sufficient aphids to resolve any preference, but not so long that a preferred prey type would be exhausted. Replicates were discarded if a larva failed to consume at least one aphid of each type. Results of choice tests were analyzed using a paired Student t test (SPSS, 1998). Adult Oviposition Assays Larvae were reared to adult stage on either BCA (C. sanguinea and H. axyridis), GCA (H. convergens), or artificial diet (C. m. fuscilabris). In an additional trial, C. sanguinea larvae were reared on an alternating diet in which they were switched to BCA after feeding for 2 days on GCA and then back to GCA after 3 days feeding on BCA. Upon eclosion, adults were confined in groups of three to five in plastic petri dishes (as above) and provisioned daily with either GCA or BCA on excised plant material. Mating occurred frequently within these groups but, once oviposition began, females (n ⫽ 10) were assigned a number and isolated to prevent the possibility of egg cannibalism by males. Females of H. convergens and C. m. fuscilabris were provided with small pieces of folded white paper towel as a substrate for oviposition; other species preferred to lay their eggs directly on the clear plastic dish or on plant material. Eggs were counted and removed daily, dishes were cleaned, and fresh aphids were provided

for a period of 4 to 6 weeks from 1st day of oviposition or until the female died. In separate experiments, females of C. sanguinea and H. axyridis were collected from the field and fed either BCA or GCA until they had oviposited for a period of 2 weeks. Fecundity data were analyzed by one-way ANOVA (SPSS, 1998). Egg clusters were held in an incubator (as above) to ascertain their viability. Females of C. m. fuscilabris reared on diet, females of C. sanguinea reared on the alternating diet, and field-collected females of H. axyridis had their diet supplemented with ice plant pollen after 1 week of oviposition, when it was clear that their egg fertility was declining. Percentages of eggs hatching were arc-sine transformed and then compared by one-way ANOVA. Egg fertility was compared before and after addition of pollen by pooling data from 3 days pre- and 3 days post-pollen addition and comparing these by ANOVA for repeated measures. Fertility and fecundity were evaluated as predictors of reproductive success by means of linear regression of mean percentage eggs hatching/female and total eggs laid/female onto total numbers of progeny. RESULTS

Coccinella septempunctata Adults of C. septempunctata collected from citrus fed readily on BCA and produced viable eggs (data not shown). Results of the developmental assays are presented in Tables 1 and 2. One C. septempunctata larvae pupated on GCA in the developmental assay but did not yield an adult. Some larvae survived when massreared on GCA and approximately 2–3% pupated successfully; half of these yielded small, but viable, adults. Larvae failing to pupate survived significantly longer on the GCA diet than on the BCA diet (Table 2). C. septempunctata adults consumed more BCA than GCA

290

J. P. MICHAUD

TABLE 2 Mean Developmental Times for Pupating Larvae of Seven Coccinellid Species and Mean Longevity of Nonsurviving Larvae Mean longevity (⫾ SEM) of larvae failing to pupate (days)

C. septempunctata C. inaequalis C. m. fuscilabris C. sanguinea H. axyridis H. convergens O. v-nigrum

Mean developmental time (⫾ SEM) of larvae pupating (days)

GCA

BCA

P

GCA

BCA

P

9.4 ⫾ 1.45 5.6 ⫾ 0.63 5.1 ⫾ 0.49 13.0 ⫾ 1.22 10.2 ⫾ 4.81 9.9 ⫾ 1.66 4.1 ⫾ 0.31

3.9 ⫾ 0.14 5.5 ⫾ 0.39 9.5 ⫾ 1.78 — 10.0 6.1 ⫾ 0.27 4.7 ⫾ 0.39

0.001 a ns a 0.01 a — ns b 0.01 a ns a

16.0 ⫾ 0.00 — — 10.7 ⫾ 0.41 20.7 ⫾ 0.83 13.5 ⫾ 0.35 —

— — 14.1 ⫾ 1.15 9.4 ⫾ 0.13 9.0 ⫾ 0.05 10.5 ⫾ 0.29 —

— — — 0.001 a 0.001 a 0.01 b —

Pupation time c (days) GCA

BCA

7.0 — — 5.5 ⫾ 0.15 6.5 ⫾ 0.23 7.4 ⫾ 0.21 —

— — 5.3 ⫾ 0.09 6.3 ⫾ 0.17 — —

Note. Larvae (n ⫽ 20) were fed from eclosion on either A. spiraecola (GCA) or T. citricida (BCA). Groups that failed a Levine test for equality of variance were compared using a Mann–Whitney U test. a Significant differences based on one-way ANOVA; ns indicates no significant difference. b Significant differences based on Mann–Whitney U test; ns indicates no significant difference. c No pupation times were significantly different.

in the choice test on the black background (Table 3), but equal numbers of each on the white background; larvae consumed equal numbers of both on white and black backgrounds.

shapen pupae after 10 –11 days. A single viable adult was obtained from numerous larvae mass-reared on GCA, suggesting a survival rate of 1–2% on this diet. Larvae of C. inaequalis expressed no preference for either aphid on either white or black backgrounds.

Coelophora inaequalis Field-collected adults of C. inaequalis produced viable eggs on a BCA diet (data not shown). Although larvae of C. inaequalis readily consumed both aphid species, none completed development on either BCA or GCA in the developmental assay (Table 1), although three larvae on the former diet formed small, mis-

Coleomegilla maculata fuscilabris Larvae of C. m. fuscilabris that failed to pupate lived significantly longer on the BCA diet than on the GCA diet (Table 2). No larvae completed development on GCA, but almost half the larvae reared on BCA yielded pupae (Table 1). Adults eclosed from 89% of these

TABLE 3 Mean Numbers (⫾ SEM) of GCA (Fourth Instar Nymphs) and BCA (Second Instar Nymphs) Consumed by Coccinellid Larvae and Adults on White and Black Backgrounds White background Species Larvae C. septempunctata C. inaequalis C. maculata fuscilabris C. sanguinea H. axyridis H. convergens Adults C. septempunctata b C. maculata fuscilabris C. sanguinea b H. axyridis b H. convergens

Black background

Test period (h)

GCA

BCA

GCA

BCA

23 24 18/24 24 22 24

1.4 ⫾ 0.20a 2.1 ⫾ 0.22a 4.9 ⫾ 0.64b a 2.1 ⫾ 0.45a 4.3 ⫾ 0.47a 2.7 ⫾ 0.34a

1.9 ⫾ 0.23a 2.1 ⫾ 0.25a 2.8 ⫾ 0.56a 2.8 ⫾ 0.30a 4.3 ⫾ 0.44a 3.2 ⫾ 0.33a

1.8 ⫾ 0.19a 3.1 ⫾ 0.39a 1.3 ⫾ 0.24a 2.3 ⫾ 0.28a 5.1 ⫾ 0.50a 2.0 ⫾ 0.32a

2.7 ⫾ 0.43a 2.9 ⫾ 0.26a 1.1 ⫾ 0.20a 2.6 ⫾ 0.49a 5.7 ⫾ 0.42a 2.1 ⫾ 0.36a

1 5 3 1 3

3.7 ⫾ 0.70a 3.9 ⫾ 0.34b 5.6 ⫾ 0.73a 4.8 ⫾ 0.79a 2.3 ⫾ 0.42a

4.0 ⫾ 0.56a 1.5 ⫾ 0.29a 4.1 ⫾ 0.73a 4.8 ⫾ 0.74a 3.3 ⫾ 0.39b

3.5 ⫾ 1.24a 4.6 ⫾ 0.45b 7.5 ⫾ 0.92b 4.5 ⫾ 0.42a 1.3 ⫾ 0.35a

6.0 ⫾ 1.30b 2.1 ⫾ 0.55a 5.3 ⫾ 0.75a 5.8 ⫾ 0.55a 4.3 ⫾ 0.49b

Note. All larvae and adults were naı¨ve unless otherwise specified. All larvae were newly eclosed first instars unless otherwise specified. Values bearing the same letter were not significantly different in a paired Student’s test, P ⬍ 0.05. a Late second instars reared on diet. b Field-collected adults.

291

DEVELOPMENT AND REPRODUCTION OF LADYBEETLES

TABLE 4 Oviposition Data (Means ⫾ SEM) for Mated Female Coccinellid Adults on Diets of A. spiraecola (GCA) and T. citricida (BCA) Mean no. eggs/day oviposition (⫾ SEM)

Mean no. days ovipositing (⫾ SEM)

Mean total fecundity (⫾ SEM)

Species

Period (days)

GCA

BCA

Pa

GCA

BCA

Pa

GCA

BCA

Pa

C. sanguinea C. m. fuscilabris b H. axyridis H. convergens

35 28 28 28

15.7 ⫾ 1.8 0.9 ⫾ 0.5 0.0 7.8 ⫾ 0.8

19.6 ⫾ 4.0 12.8 ⫾ 0.9 15.0 ⫾ 9.8 16.4 ⫾ 1.1

ns 0.001 0.001 0.001

29.3 ⫾ 2.1 1.6 ⫾ 1.0 0.0 17.1 ⫾ 2.2

28.0 ⫾ 1.6 23.1 ⫾ 1.0 17.3 ⫾ 1.6 24.6 ⫾ 0.8

ns 0.001 0.001 0.01

477.9 ⫾ 74.4 5.9 ⫾ 3.9 0.0 135.4 ⫾ 24.0

576.7 ⫾ 74.2 288.7 ⫾ 24.9 289.3 ⫾ 81.5 400.1 ⫾ 40.0

ns 0.001 0.001 0.001

Note. Cycloneda sanguinea and H. axyridis were reared on BCA, H. convergens on GCA, and C. m. fuscilabris on artificial diet. a Significant differences based on one-way ANOVA; ns indicates no significant difference. b Pollen added to diet on day 14.

pupae but were nonviable, living only 2 to 3 days and dying without mating or laying eggs. In a separate trial, larvae were transferred from artificial diet to aphids in late second instar. These larvae fed for a mean of 8.35 ⫾ 0.42 days on the GCA diet and none reached pupation, whereas 80% formed pupae on the BCA diet (F ⫽ 53.128; df ⫽ 1,37; P ⬍ 0.001) and 94% of pupae yielded viable adults which mated and laid fertile eggs. Larvae survived to pupation significantly better on pollen alone than they did on either GCA plus pollen or BCA plus pollen (F ⫽ 171.000; df ⫽ 2,57; P ⬍ 0.001; LSD, P ⬍ 0.001). When larvae were reared on GCA plus pollen, they lived only 3.45 ⫾ 0.37 days on average, compared to 3.85 ⫾ 0.39 days on BCA plus pollen (F ⫽ 0.555; df ⫽ 1, 38; P ⫽ 0.461). Ninety percent of larvae fed pollen alone completed larval development in a mean time of 16.33 ⫾ 0.44 days and 89% of pupae yielded viable adults after a mean time of 4.53 ⫾ 0.12 days. C. m. fuscilabris larvae displayed no prey preference, but adults preferred GCA on both white and black backgrounds (Table 3). The mean prereproductive period for C. m. fuscilabris females reared on artificial diet and fed BCA posteclosion (n ⫽ 16) was 6.9 ⫾ 0.27 days. Mated females (n ⫽ 11) reared on artificial diet and fed GCA posteclosion laid no eggs for their first 14 days of adult life. Pollen was added to the diet of both treatments on day 14, whereupon 3 females on the GCA plus pollen diet laid a total of 59 eggs (36, 21, and 2, respectively) over the next 21 days; only 1 of these eggs hatched. Females on the BCA diet laid a mean of 51.0 ⫾ 5 eggs over the 4 days immediately prior to the addition of pollen to their diet and 42.0 ⫾ 4 eggs over the 4 subsequent days (t ⫽ 2.165; df ⫽ 15; P ⫽ 0.047). Females on the BCA plus pollen diet oviposited on more days than females on the GCA plus pollen diet, laid more eggs per day of oviposition, and had higher total fecundity during the 4-week period (Table 4). Two females on the BCA plus pollen diet died during the period of the experiment, compared with 5 females on

the GCA plus pollen diet. The mean fertility (percentage eggs hatching) for females on the BCA plus pollen diet was only 4.17 ⫾ 1.41% (5 of 16 females laid only infertile eggs). A mean of 6.12% of eggs hatched during the first 2 weeks of oviposition, not significantly different from the 1.91% hatching during the second 2 weeks of oviposition. Linear regression of percentage eggs hatching on total female fecundity was not significant. Cycloneda sanguinea Significantly more C. sanguinea larvae yielded viable adults on the BCA diet than on the GCA diet (Table 1) and the mean developmental time of larvae was shorter on the former diet (Table 2). When larvae (n ⫽ 108) were raised on a diet alternating between GCA and BCA, 73% pupated and 86% of pupae yielded viable adults, for a net survival rate of 63%. Larvae expressed no prey preference in either choice test, but adults consumed more GCA than BCA when aphids were presented on a black background (Table 3). No significant difference in prereproductive period was found in adult females reared as larvae on BCA and fed GCA posteclosion and those fed on BCA posteclosion; the former group began ovipositing 12.1 ⫾ 1.37 days after emergence, compared with 9.3 ⫾ 1.03 days for the latter group. Females on the BCA diet had fecundity similar to that of females on the GCA diet (Table 4). Samples collected during the 4th week of oviposition revealed a mean egg hatching rate of 47.2 ⫾ 4.6% for females fed GCA (n ⫽ 44 egg masses), compared with 10.1 ⫾ 3.5% for females fed BCA (n ⫽ 46 egg masses) (F ⫽ 50.523, P ⬍ 0.001). Field-collected C. sanguinea females fed BCA (n ⫽ 6) laid significantly more eggs than did those fed GCA (n ⫽ 7) on 9 of 14 days of reproduction (Fig. 1). Whereas the fertility of females on the GCA diet remained relatively constant over the 2-week period, that of females on the BCA diet appeared to decline toward the end of the 2nd week (Fig. 2). When data on

292

J. P. MICHAUD

FIG. 1. Fecundity (A) and fertility (B) of field-collected C. sanguinea females fed either A. spiraecola (GCA) (n ⫽ 7) or T. citricida (BCA) (n ⫽ 6). Asterisks indicate days on which values differed significantly between the two groups (one-way ANOVA, P ⬍ 0.05).

percentage of eggs hatching were pooled over days 11–14, females had significantly higher fertility on the GCA diet than on the BCA diet for that period (means ⫽ 42.7 and 17.5%, respectively, F ⫽ 10.782; df ⫽ 1,47; P ⫽ 0.002). Females reared as larvae on the alternating diet had significantly greater fecundity on the BCA diet than on the GCA diet on 5 of 7 days during the 1st week of

oviposition, but not thereafter (Fig. 3). Females had similar initial fertility (Fig. 4), but from day 7 onward, females on the GCA diet had significantly higher fertility than did those on the BCA diet. The mean fertility of females on the GCA diet was significantly greater in the 2nd week of oviposition than it was in the 1st (39.4 ⫾ 4.29% vs 27.3 ⫾ 3.26%; F ⫽ 5.132; df ⫽ 1, 139; P ⫽ 0.025), whereas the opposite was true for

FIG. 2. Fecundity (A) and fertility (B) of C. sanguinea females reared as larvae on an alternating diet and fed either A. spiraecola (GCA) (n ⫽ 10) or T. citricida (BCA) (n ⫽ 10) as adults. Asterisks indicate days on which values differed significantly between the two groups (one-way ANOVA, P ⬍ 0.05).

DEVELOPMENT AND REPRODUCTION OF LADYBEETLES

FIG. 3.

293

Mean daily fertility of H. axyridis adults (n ⫽ 6) fed T. citricida.

females on the BCA diet (1.0 ⫾ 0.50% vs 10.6 ⫾ 1.87%; F ⫽ 20.484; df ⫽ 1,156; P ⬍ 0.001). The total number of eggs hatching per female displayed a significant positive correlation with total fecundity for both GCA- and BCA-fed females (r 2 ⫽ 0.705; F ⫽ 19.105; df ⫽ 1,19; P ⫽ 0.002 and r 2 ⫽ 0.451; F ⫽ 7.388; df ⫽ 1,19; P ⫽ 0.024, respectively). Mean daily fertility of females (percentage of eggs hatching) was found to be a good predictor of reproductive success as measured by total number of hatched eggs (r 2 ⫽ 0.912; df ⫽ 1,19; F ⫽ 197.02; P ⬍ 0.001), whereas total fecundity was not (r 2 ⫽ 0.12, df ⫽ 1,19; F ⫽ 0.233, P ⫽ 0.635).

15.0 ⫾ 10.0 eggs per day (Table 4) after a mean prereproductive period of 8.1 ⫾ 0.71 days. Thirty-eight egg clusters were collected during the 3rd week of oviposition to check fertility; none hatched. Field-collected females fed BCA (n ⫽ 6) initially laid eggs with high fertility, but fertility began to decline after about 1 week (Fig. 3). When pollen was added, a significantly larger proportion of eggs hatched over the following 3 days than had in the previous 3 days (F ⫽ 6.374; df ⫽ 1, 30; P ⫽ 0.017). However, this benefit appeared to be temporary and egg fertility began to decline again after 5 or 6 days.

Harmonia axyridis Larvae of H. axyridis developed with equal success on GCA and BCA, although adults developed twice as fast and were twice as heavy on the BCA diet (Tables 1 and 2). Neither larvae nor adults expressed a prey preference in choice tests on either dark or light backgrounds (Table 3). Mated female H. axyridis (n ⫽ 10) fed for 30 days posteclosion on GCA produced no eggs, whereas those fed on BCA (n ⫽ 9) laid a mean of

FIG. 4. Relative abundance of coccinellid species on aphid colonies in Florida citrus (n ⫽ 1343 observations of adult beetles).

Hippodamia convergens While some of the H. convergens larvae fed a diet of BCA reached pupation, the pupae were small and malformed and no adult beetles emerged (Table 1). Although larval developmental time was significantly longer on GCA than on BCA (Table 2), more of the larvae reached pupation and all pupae yielded viable adult beetles, although these were significantly smaller than those reared on artificial diet. There were no differences in larval developmental time, larval survival to pupation, pupation time, adult emergence, or adult weight in larvae fed on GCA from V. odoratissimum and those fed on GCA from C. sinensis. Larvae expressed no prey preference in either choice test, but adults consumed more BCA than GCA on both light and dark backgrounds (Table 3). Adult female H. convergens reared on artificial diet and transferred to an aphid diet laid more eggs per day of oviposition feeding on BCA than on GCA (26.0 ⫾ 2.25 vs 8.0 ⫾ 1.43, F ⫽ 35.62, df ⫽ 1, 13, P ⬍ 0.001) and had greater total fecundity during the 42-day period of the experiment (279.8 ⫾ 30.10 vs 51.17 ⫾ 13.22, F ⫽ 34.73, df ⫽ 1,13, P ⬍ 0.001). Similar results were obtained from adult females reared as larvae on GCA (Table 4). There was no difference between diets in the percentage of eggs hatching (means ⫽ 66.7 ⫾ 7.5 and 72.9 ⫾ 5.0% for GCA and BCA, respectively). The mean prereproductive periods were 10.7 ⫾ 1.00 days

294

J. P. MICHAUD

for females fed GCA and 12.1 ⫾ 0.34 days for females fed BCA. Olla v-nigrum No larvae of O. v-nigrum completed development on either the GCA diet or the BCA diet (Table 1). Fieldcollected adults of O. v-nigrum fed readily on BCA and GCA but produced eggs only on the former species. Four females fed BCA laid 550 eggs over a period of 20 days, with a mean hatching rate of 34.5%. After 7 days on a diet of GCA, four females had produced no eggs and were switched to a diet of BCA. Five days later they began ovipositing and produced 434 eggs in 10 days, with a hatching rate of 77.0%. DISCUSSION

Hodek (1973) reported that early instar coccinellids rarely express a preference for particular aphid prey and the results of the choice tests appeared to support this. Maternal prey selection most likely determines prey availability for larvae, at least in early instars, as suggested by Hodek (1973). This raises an interesting question for species such as C. septempuntata, C. inaequalis, C. m. fuscilabris, H. convergens, and O. v-nigrum that are able to utilize BCA for egg production, but not for larval development. Does BCA act as a “red herring” for these species, luring females into bad reproductive decisions? Okamoto and Sato (1973) and Okamoto (1974) showed that neither larvae nor adults of either C. septempuctata bruckii or H. axyridis expressed adaptive preferences for suitable prey in the presence of unsuitable alternatives. In the present study, only adults of H. convergens expressed a consistent and adaptive preference for BCA, the aphid that was most suitable for its reproduction. Conversely, C. m. fuscilabris adults preferred GCA despite better reproductive and developmental performance on BCA and adult H. axyridis did not express a preference for BCA over GCA despite the inadequacy of the latter to support egg production in this species. The preference of C. septempunctata for BCA on the dark background is difficult to explain, given that this species has been shown to select the prey which contrasts more, not less, with background coloration (Harmon et al., 1998). Only C. sanguinea and H. axyridis successfully completed development on both aphid species, with shorter developmental times and heavier adult weights resulting from the BCA diet (Tables 1 and 2). Many studies of coccinellids have found variation in larval developmental time with aphid prey (Putman, 1957; Blackman, 1967; Hukusima and Kamei, 1970; Okamoto and Sato, 1973; Michels and Behle, 1991), the more suitable prey typically yielding a shorter developmental time and heavier adults. The mean generation times for C. sanguinea (egg to adult) on BCA and GCA were

17.7 and 18.2 days, respectively, values very similar to the 18.1 days measured by Santos and Pinto (1981) for C. sanguinea feeding on T. aurantii under field conditions. Larvae of other species did not appear capable of utilizing BCA as an exclusive food source, except for C. m. fuscilabris when it was fed artificial diet in early instars. It is notable that survival of C. m. fuscilabris larvae was better on pollen alone that it was on either of the aphid plus pollen diets, suggesting that utilization of pollen was inhibited by consumption of the aphids. Although GCA was an adequate diet for the development of C. sanguinea, H. axyridis, and H. convergens, this aphid is probably an inferior prey for these coccinellid species based on the relatively low weights of the adults produced. Smirnoff et al. (1972) found that the fecundity of C. septempunctata varied with adult diet, but found no difference in egg fertility. Hukusima and Kamei (1970) measured a difference in the fecundity of H. axyridis females feeding on Myzus persicae Sulzer and Amphorophora oleraceae van der Goot and Hukusima. Ohwaki (1972) measured a difference in fecundity between diets of M. persicae and Hyperomyzus lactucae (L.). However, neither of the latter studies examined egg fertility and, in light of the results presented here, data on fecundity seems of little value without concurrent assessment of egg fertility. Although fertility and fecundity were positively correlated in C. sanguinea, fertility proved a much better predictor of reproductive success; females consuming GCA produced more viable progeny than those feeding on BCA despite laying fewer eggs. It would seem that BCA is a good protein source for ovigenesis but is deficient in some nutrient required for egg fertility. However, in the case of both C. sanguinea and H. axyridis it might be dangerous to conclude that BCA is not a suitable food on the basis of the low fertility of eggs from laboratory-reared females fed BCA exclusively, because there is no information on how females might supplement their diet in the field. Females of H. axyridis collected from BCA colonies in the field initially laid eggs of high fertility in the laboratory, although fertility declined significantly after 1 week (Fig. 3). The addition of pollen to the BCA diet at this time appeared to increase fertility, although it declined again after a number of days. H. axyridis is also known to feed on honeydew (Tedders and Schaefer, 1994) and this is another possible dietary supplement that was lacking in these experiments. When C. sanguinea was reared on a diet alternating between BCA and GCA and then fed only one species as adults, the resulting females had similar initial fertility, although fecundity was higher on the BCA diet on certain days (Fig. 3). However, differences in the fertility of females on the two diets became significant on the 7th day of reproduction (Fig. 4), indicating that larval diet affected female fertility into the 1st

DEVELOPMENT AND REPRODUCTION OF LADYBEETLES

week of reproduction. Kariluoto (1980) noted effects of larval diet on the subsequent fecundity and fertility of Adalia bipunctata (L.) adults and Phoofolo and Obrycki (1997) found an effect of larval diet on the fecundity of C. maculata females. The present data are indicative of both positive and negative effects of larval diet on fertility in C. sanguinea. The decline in fertility of females on the BCA diet could be explained by some developing nutritional deficiency on this exclusive diet, but the increase in fertility of females on the GCA diet in the 2nd week of reproduction relative to the 1st suggests some detrimental effect of consuming BCA as larvae that required more than 2 weeks of adult feeding to overcome. The large volume of eggs produced by C. sanguinea and H. axyridis on the BCA diet suggests that this aphid is a good protein source; the fact that egg fertility was low on an exclusive BCA diet indicates that this aphid is lacking in some nutrient that is essential for egg fertility in these species. Likewise, fertility of C. m. fuscilabris females was low on the BCA diet, although H. convergens females retained relatively high fertility on the same diet. It is notable that even field-collected females of C. sanguinea rarely averaged higher than 30% fertile eggs on either diet (Fig. 1). Early instar C. sanguinea larvae exhibited high levels of cannibalism of unhatched eggs and it is possible that a proportion of infertile eggs are produced as food for newly hatched larvae by females of this species. Whereas the provision of pollen to H. axyridis females appeared to increase their fertility at least temporarily, it did not diminish the declining fertility of C. sanguinea females on the BCA diet, although females readily consumed it. Females in the field may acquire some essential nutrient lacking in BCA from alternative dietary sources. If females eat varied, rather than specialized diets, then BCA may be a more suitable food for some species than the data derived from “pure” diets indicate. It is notable that these two aphid species frequently occur together in mixed colonies (Michaud, 1999; Michaud and Browning, 1999) that sometimes include A. gossypii and T. aurantii (J. P. Michaud, unpublished). More generally, it seems unlikely that adult coccinellids consume a single prey species exclusively throughout adult life under natural conditions, as pointed out by Phoofolo and Obyrycki (1997). Munyaneza and Obrycki (1998) showed that eggs of Leptinotarsa decemlineata (Say) are an adequate diet for the development of C. maculata only when larvae consume aphids as early instars. Similarly, the survival of C. m. fuscilabris larvae on BCA was much better, and resulting adults were viable, when they began feeding on this prey in late second instar, as opposed to first instar. This is in contrast to the findings of Hattingh and Samways (1992) that indicated that diet changes were deleterious for larval development in Chilocorus nigritus (F.). It appears evident that the consequences of

295

mixed diets, or prey switching, need to be assessed for both larvae and adults. In this context, the classification of aphid prey as either “essential” or “alternative” (Hodek, 1973) seems overly simplistic and laboratory studies of coccinellid performance on “simple” diets should be interpreted with caution. Aside from its apparently low nutritional value, there was no indication that the GCA diet caused any symptoms of acute toxicity in any of the beetles in this study, as reported for H. axyridis feeding on Aphis craccivora Koch from Robinia psuedo-acacia L. (Hukusima and Kamei, 1970) and Adalia bipunctata L. feeding on Megoura vicia Buckton (Blackman, 1966). Larvae failing to complete development usually fed for a number of days, but failed to grow. Larvae of species developing successfully on GCA had generally longer developmental times and produced adults of relatively low weight compared to those maturing on BCA or artificial diet or to field-collected individuals. Smith (1965) showed that the live weight of Coccinella trifasciata perplexa Mulsant was strongly influenced by dietary protein content. The fecundity of females reproducing on the GCA diet was invariably lower than that of females on the BCA diet and some species failed to produce any eggs on the former diet. These observations are all consistent with A. spiraecola representing an inferior protein source, perhaps lacking adequate levels of one or more amino acids. Coccinellid species might vary in their requirements for this amino acid during either development or ovigenesis, yielding the range of species-specific responses observed in the present study. However, the net impact of this dietary deficiency on coccinellid fitness in the field may depend on the extent to which larvae and adults are able to incorporate supplementary foods in their diet. Putman (1965) investigated the consequences of various artificial diets on the biology of three coccinellid species and found that foods inadequate for egg production could nevertheless sustain adults and increase their longevity relative to starved individuals. A. spiraecola may therefore be an important food source even for species in which it is inadequate for reproduction if it sustains adults through periods when more suitable prey is unavailable. Hodek (1973) stated that coccinellid larvae always feed on the same prey as adults. A novel finding of the present study is the apparent divergence between larvae and adults of some species with respect to optimal prey. For H. convergens, the optimal prey for maximizing larval survival (in citrus, at least) is GCA, whereas the optimal prey for maximizing reproductive success is BCA. The reverse would hold true for C. sanguinea, unless females succeed in increasing their fertility on the BCA diet through food supplements. Although larvae of O. v-nigrum, C. septempunctata, C. inaequalis, C. m. fuscilabris, and H. convergens all failed to develop successfully on BCA, adults of these species all

296

J. P. MICHAUD

laid viable eggs on a BCA diet. Larvae apparently have distinct, and probably more exacting, dietary needs than do adults, probably because of developmental requirements. Malyk and Robinson (1971) found that those coccinellid species with the best performance on cereal aphids were also the species most abundant in grain fields. Likewise, the two species of coccinellids demonstrating the best performance on citrus aphids collectively constitute 85% of adult coccinellids observed on aphid colonies in Florida citrus (Fig. 4). The present results indicate that BCA is a generally more suitable prey than GCA for these two species, and this may partially explain the noticeable increases in their abundance in citrus following the invasion of BCA in 1997 and 1998 (J. P. Michaud, unpublished). Both H. axyridis and C. sanguinea appear worthy of conservation for their potential role in reducing citrus aphid populations. Whereas H. axyridis is the larger and more voracious of the two, C. sanguinea, with its shorter generation time and ability to reproduce on both aphid species, would seem to be the best choice for augmentative releases against citrus aphids. ACKNOWLEDGMENTS I thank Integrated Biocontrol Systems, Inc. (100 Brown Street, Suite 2, Greendale, IN 47025), Kunafin (Route 1, Box 39, Quemado, TX 78877), and Predation Inc. (4445 SW 35th Terrace, Suite 310, Gainesville, FL 32608) for supplying beetles free of charge. Thanks are also due to P. Stansly for reviewing the manuscript. This work was supported by grants from USDA, APHIS, PPQ, The National Citrus Research Council, and the Florida Citrus Producers Research Advisory Council. This is Florida Agricultural Experiment Station Journal Series No. R-07197.

REFERENCES Balaraman, K., and Ramakrishnan, K. 1979. Transmission studies with strains of tristeza virus on acid lime. Z. Pflanzenkr. Pflanzen. 86, 653– 661. Blackman, R. L. 1966. Selection of aphid prey by Adalia bipunctata L. and Coccinella 7-punctata L. Ann. Appl. Biol. 59, 331–338. Blackman, R. L. 1967. The effects of different aphid foods on Adalia bipunctata L. and Coccinella 7-punctata L. Ann. Appl. Biol. 59, 207–219. Halbert, S. E. 1997. Brown citrus aphid: Current situation and prognosis. Cit. Ind. April Issue, pp. 26 –27. Harmon, J. P., Losey, J. E., and Ives, A. R. 1998. The role of vision and color in the close proximity foraging behavior of four coccinellid species. Oecologia 115, 287–292. Hattingh, V., and Samways, M. J. 1992. Prey choice and substitution in Chilocorus spp. (Coleoptera: Coccinellidae). Bull. Entomol. Res. 82, 327–334. Hodek. I. 1973. “Biology of Coccinellidae.” Junk, The Hague. Holling, C. S. 1959. Some characteristics of simple types of predation and parasitism. Can. Entomol. 91, 385–398. Holling, C. S. 1966. The functional response of invertebrate predators to prey density. Memoirs Entomol. Soc. Can. 48, 1– 86.

Hukusima, S., and Kamei, M. 1970. Effects of various species of aphids as food on development, fecundity and longevity of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Res. Bull. Fac. Agric. Gifu Univ. 29, 53– 66. Hukusima, S., and Ohwaki, T. 1972. Further notes on the feeding biology of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). Res. Bull. Fac. Agric. Gifu Univ. 33, 75– 82. Kariluoto, K. T. 1980. Survival and fecundity of Adalia bipunctata (Coleoptera, Coccinellidae) and some other predatory insect species on an artificial diet and a natural prey. Ann. Entomol. Fenn. 46, 101–106. Komazaki, S. 1988. Growth and reproduction in the first two summer generations of citrus aphids, Aphis citricola Van der Groot and Toxoptera citricidus (Kirkaldy) (Homoptera: Aphididae), under different thermal conditions. Appl. Entomol. Zool. 23, 220 –227. Malyk, M. R., and Robinson, A. G. 1971. A study of the voracity, fecundity and developmental rates of some common lady beetle predators of aphids on cereal crops in Manitoba. Man. Entomol. 5, 89 –91. Michaud, J. P. 1998. A review of the literature on the brown citrus aphid, Toxoptera citricida (Kirkaldy). Fla. Entomol. 81, 37– 61. Michaud, J. P. 1999. Sources of mortality in colonies of the brown citrus aphid, Toxoptera citricida (Kirkaldy). Biocontrol 44, 347– 367. Michaud, J. P., and Browning, H. W. 1999. Seasonal abundance of the brown citrus aphid, Toxoptera citricida (Homoptera: Aphididae) and its natural enemies in Puerto Rico. Fla. Entomol. 82, 424 – 447. Michelena, J. M., and Sanchez, A. 1997. Evolution of parasitism and beneficials on aphids in a citrus orchard. Bol. San. Veg. 23, 241– 255. Michels, G. J., and Behle, R. W. 1991. Effects of two prey species on the development of Hippodamia sinuata (Coleoptera: Coccinellidae) larvae at constant temperatures. J. Econ. Entomol. 84, 1480 – 1484. Michels, G. J., and Flanders, R. V. 1992. Larval development, aphid consumption and oviposition for five imported coccinellids at constant temperature on Russian wheat aphid and greenbugs. Southwest. Entomol. 17, 233–243. Miller, R. L. 1929. A contribution to the biology and control of the green citrus aphid, Aphis spiraecola Patch. Fla. Agric. Exp. St. Bull. 203, 431– 476. Morales, J., and Burandt, C. L., Jr. 1985. Interactions between Cycloneda sanguinea and the brown citrus aphid: Adult feeding and larval mortality. Environ. Entomol. 14, 520 –522. Munyaneza, J., and Obrycki, J. J. 1998. Development of three populations of Coleomegilla maculata (Coleoptera: Coccinellidae) feeding on eggs of Colorado potato beetle (Coleoptera: Chrysomelidae). Environ. Entomol. 27, 117–122. Okamoto, H. 1974. Laboratory studies on the food ecology of aphidophagous Coccinellids. II. A few experiments on the food preferences of the lady beetle, Harmonia axyridis Pallas and Coccinella septempunctata bruckii Mulsant (Coleoptera, Coccinellidae). Tech. Bull. Fac. Agric. Kagawa Univ. 25, 219 –224. Okamoto, H, and Sato, M. 1973. The influence of different aphids as food upon the ecological and morphological characters of the lady beetles Harmonia axyridis Pallas and Coccinella septempunctata bruckii Mulsant (Coleoptera: Coccinellidae). Kontyu 41, 342–358. Phoofolo, M. W., and Obrycki, J. J. 1997. Comparative prey suitability of Ostrinia nubilalis eggs and Acyrthosiphum pisum for Coleomegilla maculata. Biol. Control 9, 167–172. Putman, W. L. 1957. Laboratory studies on the food of some coccinellids (Coleoptera) found in Ontario peach orchards. Can. Entomol. 89, 572–579.

DEVELOPMENT AND REPRODUCTION OF LADYBEETLES Santos, G. P., and Pinto, A. C. Q. 1981. Biology of Cycloneda sanguinea and its association with aphids on mango seedlings. Pesq. Agropec. Bras. 16, 473– 476. Smirnoff, W. A., Hamalainen, M., and Markkula, M. 1972. Effect of type of food on fecundity in Coccinella septempunctata L. (Coleoptera: Coccinellidae). Ann. Entomol. Fenn. 38, 195–199. Smith, B. C. 1965. Effects of food on the longevity, fecundity and development of adult coccinellids (Coleoptera: Coccinellidae). Can Entomol. 97, 910 –919. Solomon, M. E. 1949. The natural control of animal populations. J. Anim. Ecol. 18, 1–35. SPSS. 1998. SPSS version 8.0. SPSS Inc., Chicago, IL. Tao, C.C., and Chiu, C. 1971. Biological control of citrus, vegetable

297

and tobacco aphids. Spec. Publ. Taiwan Agric. Res. Inst. 10, 1–110. Tedders, W. L., and Schaefer, P. W. 1994. Release and establishment of Harmonia axyridis (Coleoptera: Coccinellidae) in the southeastern United States. Entomol. News 105, 228 –243. Yokomi, R. K., and Damsteegt, V. C. 1991. Comparison of citrus tristeza virus transmission efficacy by Toxoptera citricidus and Aphis gossypii. In “Proceedings, Aphid–Plant Interactions: Populations to Molecules” (D. C. Peters, J. A. Webster, and C. S. Choubler, Eds.), pp. 229 –241. Stillwater, OK. Yokomi, R. K., and Tang, Y. Q. 1996. A survey of parasitoids of brown citrus aphid (Homoptera: Aphididae) in Puerto Rico. Biol. Control 6, 222–225.